Linker For Activation Of T-cells Research Articles

Mutual regulation between coupled protein condensates and ordered membrane domains.

Downstream of T-cell receptor ligation, biomolecular condensates are produced via the multivalent binding between Linker for Activation of T-cells (LAT) and its effectors (Grb2 and Sos1) to facilitate T-cell activation. LAT is a transmembrane protein known to be recruited to ordered lipid domains, suggesting potential coupling between protein cytoplasmic condensates and lateral lipid-based phase-separation and organization. We report evidence demonstrating thermodynamic coupling between lipid- and protein-based phase separation. In model membranes, condensates formed on phase-separating membranes template ordered lipid domains and induce phase separation. Conversely, lipid phase separation stabilizes protein condensate formation suggesting strong coupling between lipid- and protein-based phase separation. Furthermore, experiments in T-cells show that LAT/Grb2/Sos1 condensates recruit ordered domain lipids and proteins in living cells. We observed that crosslinking of a membrane component that prefers ordered domains (i.e. the endogenous GPI-anchored protein Thy1) stabilizes lipid phase separation and templates condensate formation. Finally, we tested whether condensate-domain coupling is necessary for T-cell activation by decoupling raft domains from condensates through either mutation of the transmembrane domain to modulate its raft affinity or mutation of residues necessary for condensate formation. We found that the coupling of cytoplasmic condensate formation to lipid rafts is essential for T-cell activation. Thus, we conclude that the thermodynamic coupling between LAT/Grb2/Sos1 cytoplasmic condensates and lipid rafts can cooperatively and functionally reorganize the membrane to facilitate T-cell activation.

A possible way to prevent the progression of bone lesions in multiple myeloma via Src-homology-region-2-domain-containing-phosphatase-1 activation.

On the basis of our recent findings, in which multiple receptor-mediated mast cell functions are regulated via a common signaling cascade, we posit that the formation and functioning of osteoclasts are also controlled by a similar common mechanism. These cells are derived from the same granulocyte/monocyte progenitors and share multiple receptors except those that are cell-specific. In both types of cells, all known receptors reside in lipid rafts, form multiprotein complexes with recruited signaling molecules, and are internalized upon receptor engagement. Signal transduction proceeds in a chain of protein phosphorylations, where adaptor protein LAT (linker-for-activation-of-T-cells) plays a central role. The key kinase that associates LAT phosphorylation and lipid raft internalization is Syk (spleen-tyrosine-kinase) and/or an Src-family-kinase, most probably Lck (lymphocyte-specific-protein-tyrosine-kinase). Dephosphorylation of phosphorylated Syk and Lck by activated SHP-1 (Src-homology-region-2-domain-containing-phosphatase-1) terminates the signal transduction and endocytosis of receptors, resulting in inhibition of osteoclast differentiation and other functions. In malignant plasma cells (MM cells) too, SHP-1 plays a similar indispensable role in controlling signal transduction required for survival and proliferation, though BLNK (B-cell-linker-protein), a functional equivalent of LAT and SLP-76 (SH2-domain-containing-leukocyte-protein-of-76-kDa) in B cells, is used instead of LAT. In both osteoclasts and MM cells, therefore, activated SHP-1 acts negatively in receptor-mediated cellular functions. In osteoblasts, however, activated SHP-1 promotes differentiation, osteocalcin generation, and mineralization by preventing both downregulation of transcription factors, such as Ostrix and Runx2, and degradation of β-catenin required for activation of the transcription factors. SHP-1 is activated by tyrosine phosphorylationand micromolar doses (M-dose) of CCRI-ligand-induced SHP-1 activation. Small molecular compounds, such as A770041, Sorafenib, Nitedanib, and Dovitinib, relieve the autoinhibitory conformation. Activation of SHP-1 by M-dose CCRI ligands or the compounds described may prevent the progression of bone lesions in MM.

ERM-Dependent Assembly of T Cell Receptor Signaling and Co-stimulatory Molecules on Microvilli prior to Activation

T cell surfaces are covered with microvilli, actin-rich and flexible protrusions. We use super-resolution microscopy to show that ≥90% of T cell receptor (TCR) complex molecules TCRαβ and TCRζ, as well as the co-receptor CD4 (cluster of differentiation 4) and the co-stimulatory molecule CD2, reside on microvilli of resting human Tcells. Furthermore, TCR proximal signaling molecules involved in the initial stages of the immune response, including the protein tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase) and the key adaptor LAT (linker for activation of Tcells), are also enriched on microvilli. Notably, phosphorylated proteins of the ERM (ezrin, radixin, and moesin) family colocalize with TCRαβ as well as with actin filaments, implying a role for one or more ERMs in linking the TCR complex to the actin cytoskeleton within microvilli. Our results establish microvilli as key signaling hubs, in which the TCR complex and its proximal signaling molecules and adaptors are preassembled prior to activation in an ERM-dependent manner, facilitating initial antigen sensing.

Analysis of T-Cell Receptor Beta-Constant Region Expression for Rapid Assessment of T-Cell Clonality

IntroductionT-cell malignancies are heterogenous disorders, representing approximately 10-15% of cases of non-Hodgkin's lymphoma and 15% of acute lymphoblastic leukaemia. Diagnosis may be complicated both by the fact that non-malignant oligoclonal T-cell proliferations often mimic T-cell cancers, and due to the lack of consistent markers of malignancy which discriminate healthy and malignant T-cells. B-cell lymphoproliferations, by contrast, can be reliably identified as clonal by confirmation of restricted kappa or lambda light chain expression. We have recently described an analogous method for evaluation of T-cell clonality, by evaluating expression of one of two virtually identical and mutually exclusive alleles at the T-cell receptor beta constant region: TRBC1 or TRBC2. We demonstrated that approximately 35% of normal T-cells and T-cell malignancies expressed TRBC1, using a TRBC1-specific antibody suitable for use in fresh/ frozen tissues. We have now developed a novel antibody with TRBC2 specificity, which recognises an intracellular, non-surface exposed epitope and can be used in both fresh and formalin-fixed paraffin-embedded (FFPE) tissues. Here we demonstrate TRBC1/2 assessment can be used to identify T-cell clonality using immunohistochemistry on FFPE tissues, and confirm our results using reference methods including Sanger and massively parallel sequencing.Methods/ ResultsTo generate anti-TRBC2 antibody, we immunised New Zealand rabbits with a peptide comprising the intracellular portion of the TCR beta-2 chain (VKRKDSRG). Protein A-purified immunoglobulin from the sera of immunised rabbits was tested for reactivity to TRBC1 (VKRKDF) and TRBC2 peptides by ELISA. Strong TRBC2-specificity with limited TRBC1 binding was seen. Immunoglobulin was then depleted of residual TRBC1 reactivity by depletion on TRBC1 peptide-coated agarose beads. Following this, no reactivity to TRBC1 peptide could be detected by ELISA.We then stained fresh frozen and FFPE samples of pelleted TRBC1 and TRBC2 cell lines with the novel, polyclonal anti-TRBC2 antibody. We confirmed that anti-TRBC2 antibody stained only TRBC2 and not TRBC1 cell lines, and that the antibody performed equivalently in fresh or FFPE preparations. We then showed that in a range of normal tissues, including tonsil, spleen and others, a staining pattern restricted to only a proportion of T-cells was seen. In bone marrow sections, some staining of megakaryocytes was also seen, as has been previously demonstrated for other T-cell markers such as linker for activation of T-cells (LAT). No other non-T-cell staining was noted.We obtained paired FFPE and frozen samples of several TCR+ T-cell malignancies. We performed staining for TRBC1 and TRBC2 on both frozen and FFPE samples: TRBC1 staining was performed only on frozen samples. In addition, we extracted DNA from these tissues and performed PCR for TCR beta VDJ rearrangement using standard BIOMED protocols. Clonal proliferations were identified by heteroduplex gel analysis and capillary electrophoresis (Sanger) sequencing was performed on dominant bands. In addition, pooled PCR products were subjected to massively parallel sequencing to quantify clone frequency and to confirm VDJ identity. We demonstrated mutual exclusivity between samples staining positive for TRBC1 and TRBC2, and concordance between TRBC1/2 status identified by Sanger sequencing, NGS and antibody-based methods.Further, in a number of TCR+ malignancies, non-T-cell haematological tumours and reactive lymphoproliferations, where only FFPE material was available, we used anti-TRBC2 antibody to evaluate TRBC2 expression. We demonstrated that in non-T cell tumours, a limited pattern of staining was seen, restricted to a proportion of infiltrating normal T-cells only. T-cell tumours, by contrast, were uniformly TRBC2-negative or positive, across a range of entities. 18/29 (62%) of T-cell neoplasms were TRBC2-positive.ConclusionsWe have generated a new TRBC2-specific polyclonal reagent suitable for use in FFPE tissues, which displays minimal cross-reactivity against other cell types. A monoclonal reagent is in preparation. We suggest that TRBC1/2 assessment can thus be used to rapidly identify clonal T-cell lymphoproliferations and may be a helpful addition to the diagnostic haematopathology toolkit. DisclosuresMaciocia:Autolus: Equity Ownership, Patents & Royalties: UCLB. Neves:Autolus Ltd: Employment. Onuoha:Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Pule:UCLB: Patents & Royalties; Autolus: Employment, Equity Ownership.

SYK expression in monomorphic epitheliotropic intestinal T-cell lymphoma

Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), formerly known as type II enteropathy associated T-cell lymphoma (type II EATL), is a rare, aggressive primary intestinal T-cell lymphoma with a poor prognosis and an incompletely understood pathogenesis. We collected 40 cases of MEITL and 27 cases of EATL, formerly known as type I EATL, and comparatively investigated the T-cell receptor (TCR) itself and associated signaling molecules using immunohistochemistry, amplicon deep sequencing and bisulfite pyrosequencing. The TCR showed both an αβ-T-cell origin (30%) and a γδ-T-cell derivation (55%) resulting in a predominant positive TCR phenotype in MEITL compared with the mainly silent TCR phenotype in EATL (65%). The immunohistochemical expression of the spleen tyrosine kinase (SYK) turned out to be a distinctive feature of MEITL (95%) compared with EATL (0%). Aberrant SYK overexpression in MEITL is likely caused by hypomethylation of the SYK promoter, while no common mutations in the SYK gene or in its promoter could be detected. Using amplicon deep sequencing, mutations in DNMT3A, IDH2, and TET2 were infrequent events in MEITL and EATL. Immunohistochemical expression of linker for activation of T-cells (LAT) subdivided MEITL into a LAT expressing subset (33%) and a LAT silent subset (67%) with a potentially earlier disease onset in LAT-positive MEITL.

A role for GPI-CD59 in promoting T-cell signal transduction via LAT.

Cluster of differentiation 59 (CD59) is a glycosylphosphatidylinositol-anchored protein. Cross-linking of CD59 with specific monoclonal antibodies can cause a series of intracellular signal transduction events. However, the underlying molecular mechanisms are poorly understood. Linker for activation of T-cells (LAT) is a crucial adaptor protein in T-cell signaling, and its phosphorylation and palmitoylation are essential for its localization and function. In a previous study by the present authors, it was demonstrated that CD59 may be responsible for LAT palmitoylation, thereby regulating T-cell signal transduction. The present study detected the co-localization of LAT and CD59 in lipid rafts by transfecting Jurkat cells with lentivirus vectors carrying the LAT-enhanced green fluorescent protein fusion protein. In addition, LAT and CD59 were shown to have a synergistic effect on the proliferation of Jurkat cells. The results also indicated that CD59 may transfer the palmitate group from phosphatidylinositol to LAT to form LAT palmitate, which then localizes to lipid rafts to regulate T-cell activation. The results of the present study provided novel insights into the role of CD59 in T-cell signal transduction.

The FcεRI Signaling Cascade and Integrin Trafficking Converge at Patterned Ligand Surfaces.

We examined the spatial targeting of early and downstream signaling mediated by the IgE receptor (FcεRI) in RBL mast cells utilizing surface-patterned 2,4 dinitrophenyl (DNP) ligands. Micron-sized features of DNP are presented as densely immobilized conjugates of bovine serum albumin (DNP-BSA) or mobile in a supported lipid bilayer (DNP-SLB). Although soluble anti-DNP IgE binds uniformly across features for both pattern types, IgE bound to FcεRI on cells shows distinctive distributions: uniform for DNP-SLB and edge-concentrated for DNP-BSA. These distributions of IgE-FcεRI propagate to the spatial recruitment of early signaling proteins, including spleen tyrosine kinase (Syk), linker for activation of T cells (LAT), and activated phospholipase C gamma 1 (PLCγ1), which all localize with engaged receptors. We found stimulated polymerization of F-actin is not required for Syk recruitment but is progressively involved in the recruitment of LAT and PLCγ1. We further found β1- and β3-integrins colocalize with IgE-FcεRI at patterned ligand surfaces as cells spread. This recruitment corresponds to directed exocytosis of recycling endosomes (REs) containing these integrins and their fibronectin ligand. Together, our results show targeting of signaling components, including integrins, to regions of clustered IgE-FcεRI in processes that depend on stimulated actin polymerization and outward trafficking of REs.

Elucidation of a Raft-Partitioning Motif in Transmembrane Proteins

Eukaryotic plasma membranes (PMs) are believed to possess lateral lipid domains termed lipid rafts, which form functional platforms for membrane sorting and cell signalling. The function of these domains depends on their selective recruitment of specific membrane proteins. However the underlying structural features governing protein partition into lipid rafts remain unknown. In live cells, the nanoscopic size and subsecond lifetime of rafts presents great difficulty for measuring their properties and composition. Intact PMs isolated as Giant Plasma Membrane Vesicles (GPMVs) phase separate into two microscopic, stable lipid domains, with one of these domains possessing greater lipid order, lower diffusivity and enriching for canonical raft components. This microscopic raft phase separation thus presents an optimal system for measuring protein partitioning between coexisting membrane domains. Using GPMVs, we demonstrate that a protein's transmembrane domain (TMD) is a central determinant of raft affinity. A major factor governing raft partition was the length of the TMD, with longer TMDs preferring the thicker raft domains. Further, by systematic mutation of the TMD of LAT (Linker for activation of T-cells), we were able to determine amino acid sequences which are necessary and sufficient for raft partitioning and which highly resemble the α-helix oligomerization motif GxxxG. This resemblance suggested that TMD oligomerization is the structural basis for the discovered raft-partitioning motif. We confirmed this hypothesis by measure sequence-dependent TMD oligomerization by fluorescence lifetime microscopy (FLIM) combined with Forster resonance energy transfer (FRET). Finally, we demonstrate the biological significance of our observations by showing that raft partitioning determines sorting between organellar membranes. For the entire panel of TMD-variants, we observed a strong quantitative relationship between raft association and sorting to the plasma membrane, with non-raft mutants being sorted into the lysosomes for degradation.

Membrane raft association is a determinant of plasma membrane localization.

The lipid raft hypothesis proposes lateral domains driven by preferential interactions between sterols, sphingolipids, and specific proteins as a central mechanism for the regulation of membrane structure and function; however, experimental limitations in defining raft composition and properties have prevented unequivocal demonstration of their functional relevance. Here, we establish a quantitative, functional relationship between raft association and subcellular protein sorting. By systematic mutation of the transmembrane and juxtamembrane domains of a model transmembrane protein, linker for activation of T-cells (LAT), we generated a panel of variants possessing a range of raft affinities. These mutations revealed palmitoylation, transmembrane domain length, and transmembrane sequence to be critical determinants of membrane raft association. Moreover, plasma membrane (PM) localization was strictly dependent on raft partitioning across the entire panel of unrelated mutants, suggesting that raft association is necessary and sufficient for PM sorting of LAT. Abrogation of raft partitioning led to mistargeting to late endosomes/lysosomes because of a failure to recycle from early endosomes. These findings identify structural determinants of raft association and validate lipid-driven domain formation as a mechanism for endosomal protein sorting.

FcεRI Signal Propagation is Regulated through Transient Binding of Syk

The high affinity IgE receptor, FceRI, serves as the primary immunoreceptor on mast cells and basophils.Crosslinking of IgE-bound FceRI via multivalent antigen initiates Src family kinase mediated phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs), recruitment of the kinase Syk, and propagation of signaling through the scaffolding protein Linker for Activation of T-cells (LAT). While the sequence of these signaling events has been well studied using biochemical techniques, the biophysical mechanisms that regulate FceRI signaling are unclear because protein activation and dynamic protein-protein interactions are difficult to quantify on living cells. Using two-color single molecule imaging in live cells, we quantify how FceRI aggregation influences SYK recruitment and LAT mobility.We imaged the basal surface of RBL-2H3 cells expressing GFP-Syk using TIRF microscopy and upon crosslinking of FceRI observed an increase in the membrane localization of GFP-Syk. The formation of GFP-Syk clusters directly co-localized with crosslinked FceRI and the intensity of these clusters increased linearly with FceRI aggregate size. Interestingly, while ensemble measurements showed that FceRI and GFP-Syk were co-localized over minutes, single molecule imaging revealed that GFP-Syk clusters are in fact maintained through a continuous exchange of transiently bound GFP-Syk. We quantify the residency time of individual GFP-Syk molecules at FceRI aggregates and explore the relationship between FceRI aggregate size and Syk residency time. The observed transient GFP-Syk binding supports the hypothesis that phosphorylated SYK moves from FceRI clusters to bind and activate LAT. To explore this mechanism further, we quantify the influence of FceRI crosslinking on the spatiotemporal distribution of LAT. We have generated a Fluorogen Activating Protein (FAP) -tagged LAT construct and take advantage of the far-red emission of the FAP probe to simultaneously track FAP-LAT mobility with respect to FceRI/SYK patches.

CD25 and CD69 induction by α4β1 outside-in signalling requires TCR early signalling complex proteins

Distinct signalling pathways producing diverse cellular outcomes can utilize similar subsets of proteins. For example, proteins from the TCR (T-cell receptor) ESC (early signalling complex) are also involved in interferon-α receptor signalling. Defining the mechanism for how these proteins function within a given pathway is important in understanding the integration and communication of signalling networks with one another. We investigated the contributions of the TCR ESC proteins Lck (lymphocyte-specific kinase), ZAP-70 (ζ-chain-associated protein of 70 kDa), Vav1, SLP-76 [SH2 (Src homology 2)-domain-containing leukocyte protein of 76 kDa] and LAT (linker for activation of T-cells) to integrin outside-in signalling in human T-cells. Lck, ZAP-70, SLP-76, Vav1 and LAT were activated by α4β1 outside-in signalling, but in a manner different from TCR signalling. TCR stimulation recruits ESC proteins to activate the mitogen-activated protein kinase ERK (extracellular-signal-regulated kinase). α4β1 outside-in-mediated ERK activation did not require TCR ESC proteins. However, α4β1 outside-in signalling induced CD25 and co-stimulated CD69 and this was dependent on TCR ESC proteins. TCR and α4β1 outside-in signalling are integrated through the common use of TCR ESC proteins; however, these proteins display functionally distinct roles in these pathways. These novel insights into the cross-talk between integrin outside-in and TCR signalling pathways are highly relevant to the development of therapeutic strategies to overcome disease associated with T-cell deregulation.

Turnabout is Fair Play: Mutual Regulation of Palmitoylation and Raft Partitioning

The physical basis of protein partitioning into lipid rafts remains an outstanding question in membrane biology that has previously been addressed only through indirect techniques involving differential solubilization by non‐ionic detergents. We have used Giant Plasma Membrane Vesicles (GPMVs), a plasma membrane model system that phase separates to include an ordered phase enriching for raft constituents, to analyze the structural determinants for raft partitioning of the transmembrane protein Linker for Activation of T‐cells (LAT). LAT enrichment in the raft phase was dependent on palmitoylation at two juxtamembrane cysteines. This palmitoylation requirement for raft association was also shown for the majority of integral raft proteins. Independently, systematic mutations that disrupted raft partitioning showed a causal relationship between raft association and plasma membrane (PM) localization, with non‐raft mutants mis‐sorted to late endosomes and depalmitoylated by the lysosomal protein thioesterase PPT1. These findings implicate lipid‐driven phase separation as one mechanism for protein sorting at the late stage of the secretory pathway and suggest mutual regulation of palmitoylation and raft partitioning of LAT.

Single Particle Tracking using Fluorogen Activating Peptides to Investigate FcεRI Signaling Dynamics

The high affinity IgE receptor, FceRI, serves as the primary immunoreceptor on mast cells and basophils. Cross-linking of IgE-bound FceRI via multivalent antigen initiates a signaling cascade that ultimately results in the release of key mediators for an allergic response. Priming with IgE has been traditionally thought of as a passive event, yet certain forms of IgE termed ‘cytokinergic’ IgEs (cIgE) have been shown to induce activation independent of antigen. The mechanism of action of cIgEs remains unclear. To determine whether changes in receptor mobility are associated with cIgE activation, we have developed a fluorogen activating peptide (FAP)-tagged FceRI ɣ-subunit for single particle tracking. FAPs are genetically expressible tags that bind an exogenous fluorogen dye. The titration of the fluorogen dye allows the labeling density to be adjusted at the time of imaging without the need to photo-bleach or photo-activate a population of dye molecules. We find that even in the presence of concentrations of cIgE that induce robust signaling, FceRI remains highly mobile.We take advantage of the FAP system to additionally investigate the mobility of the downstream scaffolding protein Linker for Activation of T-cells (LAT). The mechanism through which FceRI crosslinking propagates signaling to LAT remains a topic of debate as LAT has been previously shown to form clusters segregated from FceRI after activation. However, these studies were carried out using high concentrations of multivalent allergen that resulted in large immobile FceRI aggregates. The far-red emission of the MG2P fluorogen allows for simultaneous tracking of FAP-LAT mobility with respect to FceRI labeled with Alexa488-IgE. Using FAP-tagged LAT, we investigate how LAT mobility and distribution change over a range of activating conditions, in which FceRI goes from mobile to immobile crosslinked aggregates.

T-cell signaling and immunopathologies

The understanding of the factors responsible for T-cellsignaling has developed enormously over the past twodecades. Our initial discovery of the CD4- and CD8-p56lckcomplexes and their ability to phosphorylate the T-cellreceptor (TCR) complex led to the discovery of a secondprotein-tyrosine kinase ZAP-70 and an array of adaptorproteins such as LAT and adhesion and degranulation-promoting adaptor protein (ADAP) that are responsible forintegrating TCR signaling events. These pathways are inturn modified by phosphatases such as LYP (PTPN22), E3ligases of the ubiquitin pathway such as Cbl-b and Itch, andco-receptor modules such as signaling lymphocyte activa-tion molecule (SLAM). TCR signals then determine thefunction of T-cells subsets such as regulatory T-cells (Tregs)and follicular helper T-cells (Tfh) in their regulation ofimmune function and autoimmunity. This volume of Semi-nars in Immunopathology provides an up-to-date review ofthe current status of mutations in the TCR signaling cascadeand T-cell subsets that account for immunodeficiencies andautoimmune pathologies.During the past two decades, the field of T-cell signalinghas developed in leaps and bounds. Our initial discovery ofthe CD4- and CD8-p56lck complexes identified theinitiators of the TCR tyrosine phosphorylation cascade inT-cells (i.e., referred to as the “TCR signaling paradigm”)[1–3]. p56lck phosphorylates the antigen-receptor complex[2, 4] in a cascade that leads to the recruitment of anotherkinase termed ZAP-70 to immunoreceptor tyrosine-basedactivation motifs (ITAMs; YxxL x6–8 YxxL) on the CD3and zeta chains [5]. These observations combined withavailability of antiphosphotyrosine-specific antibodies ledto the identification of an array of p56lck, p59fyn, andZAP-70 targets that have served as the mainstay of researchon T-cell activation over the past two decades. Whilep56lck can phosphorylate ITAMs, related src kinase p59fynpreferentially phosphorylates ADAP [6, 7], and ZAP-70targets linker for activation of T-cells (LAT) and 76-kDa srchomology 2 domain-containing leukocyte phosphoprotein(SLP-76) [8, 9]. From this work evolved the identificationof so-called immune cell specific “adaptor proteins” or“molecular scaffolds” that possess discrete moleculardomains or binding sites for the generation of complexes[10–12]. These complexes of proteins integrate TcR signalsfor downstream effecter functions such as cytokine produc-tion [11]. For example, LAT has multiple sites for bindingto phospholipase Cγ1 (PLCγ1), GADS, and growth factorreceptor-bound protein 2 [13, 14], while SLP-76 constitu-tively binds to GADS and has binding sites for Vav1, IL2-inducible T-cell kinase, PLCγ1, 14-3-3, and an SH2domain that binds to ADAP and hematopoietic progenitorkinase 1 [8, 10]. At the same time, key protein-tyrosinephosphatases such as CD45, SHP-1, and LYP have beenuncovered that amplify or dampen the function of thesemolecules by dephosphorylating proteins [15]. Over-arching on this scheme is the ubiquitination pathway, amechanism that controls the expression of signalingproteins [16, 17]. Ubiquitination is controlled by an arrayof E1-E3 ligases that tag proteins for degradation byproteasomes. In addition, co-receptors such as CD28 canamplify the immune response [18, 19]. In this context,SLAM uses a unique recognition mode of tyrosinephosphorylation to induce co-signals [20, 21]. ThesecomponentsinturncanregulatethedevelopmentandfunctionofT-cellsubsets.OfparticularnoteareTRegsandtheircrucialrole in regulating autoimmunity [22]. Given these develop-

Specific Pharmacological Targeting of the Syk Kinase Activity in Platelets: A Novel, Safe Anti-Thrombotic Strategy

Studies of the Syk −/− mouse have implicated spleen tyrosine kinase (Syk), a signaling protein with both kinase and scaffolding activities, in platelet signaling following engagement of GPVI and αIIbβ3 by collagen and fibrinogen, respectively. The present study was designed to determine whether specific inhibition of the kinase activity of Syk, without targeting the Syk scaffolding function, affected in vivo arterial thrombosis. In preliminary experiments, blood from wild-type and Syk−/− mice was perfused through collagen-coated capillaries under arterial shear rates to study ex vivo thrombosis. While blood from wild-type mice formed robust thrombi (37±4.7 μm3/μm2), none was observed in Syk−/− mice. Thrombi intermediate in size (16±3.9 μm3/μm2) developed in Syk+/− mice. To achieve specific pharmacological targeting of the kinase activity of Syk, P142-76, a potent (IC50 = 4 nM) and selective Syk kinase inhibitor was utilized. P142-76 was screened against a broad panel of 139 purified kinases at 50 nM. While Syk kinase was inhibited by 92%, all other kinases retained more than 70% of their activity. In washed human platelets, P142-76 inhibited convulxin (CVX)-induced phosphorylation of LAT (linker for activation of T-cells; IC50 = 111 nM) and intracellular calcium increases (IC50 = 31 nM). The GPVI/Syk-specificity of P142-76 activity was confirmed by its inability to inhibit intracellular calcium increases induced by the PAR1 thrombin receptor agonist TRAP. P142-76 also inhibited CVX-induced aggregation of both human washed platelets (IC50 = 87 nM) and platelet-rich plasma (IC50 = 2.5 μM). Considering the controversial data in respect to the participation of GPVI in arterial thrombosis in murine models, the dependence of arterial thrombosis on Syk function was studied in vivo in pigs. Cross-species activity of P142-76 was confirmed in vitro (CVX-induced PRP aggregation IC50= 350 nM; 5 μM P142-76 completely inhibited thrombosis triggered by collagen in the perfusion chamber assay). At a plasma concentration which abolished ex vivo CVX-induced but not ADP-induced pig platelet aggregation, P142-76 significantly inhibited the deposition of [111In]-labeled platelets in a carotid artery crush swine thrombosis model, without compromising primary hemostasis.% aggregationSwine (n=3)Platelet Deposition % inhibitionPlasma Conc (ng/ml)Bleed Time (min)Activated Clotting Time (sec)ADP (20 μM)CVX (250 ng/ml)Control Artery003±0.9133±22100100Treated Artery76±6.51343±3043.5±0.3130±131000To clarify further the contribution of the kinase activity of Syk to arterial thrombosis, effects of P142-76 on human blood were evaluated in real time in the collagen-coated perfusion chamber. Low concentrations of P142-76 (0.3 μM) affected thrombus stability, while increasing concentrations (1–5 μM) delayed and then completely inhibited thrombus formation. Furthermore, P142-76 destabilized pre-formed thrombi, indicating a critical role for Syk in conferring strength to platelet-platelet interactions, i.e. αIIbβ3-mediated cohesion. Our data indicate that the kinase activity of Syk acts in arterial thrombosis through at least two distinct mechanisms. First, Syk kinase confers stability to platelet-platelet interactions downstream of αIIbβ3. Second, it initiates thrombus formation on collagen surfaces. This dual activity of the kinase activity of Syk makes it a preferred target for inhibition of arterial thrombosis, as it does not compromise primary hemostasis.

A novel ZAP-70 dependent FRET based biosensor reveals kinase activity at both the immunological synapse and the antisynapse.

Many hypotheses attempting to explain the speed and sensitivity with which a T-cell discriminates the antigens it encounters include a notion of relative spatial and temporal control of particular biochemical steps involved in the process. An essential step in T-cell receptor (TCR) mediated signalling is the activation of the protein tyrosine kinase ZAP-70. ZAP-70 is recruited to the TCR upon receptor engagement and, once activated, is responsible for the phosphorylation of the protein adaptor, Linker for Activation of T-cells, or LAT. LAT phosphorylation results in the recruitment of a signalosome including PLCγ1, Grb2/SOS, GADS and SLP-76. In order to examine the real time spatial and temporal evolution of ZAP-70 activity following TCR engagement in the immune synapse, we have developed ROZA, a novel FRET-based biosensor whose function is dependent upon ZAP-70 activity. This new probe not only provides a measurement of the kinetics of ZAP-70 activity, but also reveals the subcellular localization of the activity as well. Unexpectedly, ZAP-70 dependent FRET was observed not only at the T-cell -APC interface, but also at the opposite pole of the cell or “antisynapse”.

LAT Plays Important Role in Thymocyte Survival and Mature T Cell Activation (87.35)

Abstract LAT (Linker for Activation of T-cells) is a membrane-associated adaptor protein that plays a critical role in linking T-cell receptor engagement to activation of its downstream signaling events. Upon tyrosine phosphorylation, LAT binds Grb2, Gads, and PLC-gamma1. Previous studies show that LAT is essential in thymocyte development as LAT-deficient mice completely lack mature T cells. In addition, LAT-deficient Jurkat cells have a severe defect in TCR-mediated signaling. In order to investigate the role of LAT in T cell activation, survival, and homeostasis, mice in which the LAT gene can be inducibly deleted from thymocytes and mature T cells by injection of tamoxifen were generated. Our data showed that tamoxifen treatment of these mice led to a dramatic loss of thymocytes, especially DP thymocytes. Deletion of LAT in mature T cells affected TCR-mediated signaling, IL-2 production, and proliferation. These data suggested that LAT plays an important role in thymocyte survival and TCR-mediated signaling in mature T cells.

A Network Analysis of Changes in Molecular Interactions in Cellular Signaling

Multiprotein complexes play an essential role in the propagation and integration of cellular signals. However, systems level analyses of signaling-dependent changes in the pattern of molecular interactions are still missing. Signaling in T-lymphocytes is one prominent example in which multiprotein complexes orchestrate signal transduction. We implemented peptide microarrays comprising a set of interaction motifs of signaling proteins for network-based analyses of signaling-dependent changes in molecular interactions. Lysates of resting or stimulated cells were incubated on these arrays, and the binding of signaling proteins was detected by immunofluorescence. Signaling-dependent complex formation led to changes of signals on the microarrays in two ways. 1) Masking of a binding site of a signaling protein for a peptide on the array resulted in a signal decrease. 2) Interaction of a protein with a second protein, which in turn binds to a peptide on the array, resulted in a signal increase for the first protein. Dissipation of complexes led to the reverse changes. Competition with peptides corresponding to interaction motifs provided detailed information on the architecture of complexes; lack of individual signaling proteins revealed the functional interdependence of interactions in the network. We show that complex formation through phosphorylation of the scaffolding protein LAT (linker for activation of T-cells) acted as a signal amplifier. PLCgamma1 deficiency increased the resting state levels of LAT-dependent complexes and augmented the recruitment of the phosphatase SHPTP2 into complexes. For the analysis of signaling networks, the parallel detection of changes in interactions enabled the identification of functional interdependencies with minimum a priori knowledge.

EphrinB1 Is Essential in T-cell-T-cell Co-operation during T-cell Activation

Eph kinases are the largest family of receptor tyrosine kinases, and their ligands are ephrins (EFNs), which are also cell surface molecules. We have very limited knowledge about the expression and function of these kinases and their ligands in the immune system. In this study we investigated the effect of EFNB1 on mouse T-cells. EFNB1 and the Eph kinases it interacts with (collectively called EFNB1 receptors (EFNB1R)) were expressed on T-cells, B cells, and monocytes/macrophages. Some T-cells were double positive for EFNB1 and EFBB1R. Solid phase EFNB1 in the presence of suboptimal TCR ligation augmented T-cell responses in terms interferon-gamma secretion, proliferation, and cytotoxic T lymphocyte activity but not interleukin-2 production. After T-cell receptor (TCR) ligation, EFNB1R congregated to TCR caps, and then both of them translocated to raft caps. This provides a morphological basis for EFNB1R to enhance TCR signaling. Further downstream of the signaling pathway, EFNB1R stimulation led to increased LAT (linker for activation of T-cells) phosphorylation and p44/42 and p38 MAPK activation. Similar to CD28 costimulation, EFNB1R costimulation was insensitive to cyclosporin A inhibition. On the other hand, unlike the former, EFNB1R costimulation failed to activate Akt, which is essential in triggering interleukin-2 production. Our study suggests that EFNB1 is pivotal in T-cell-T-cell costimulation and in reducing T-cell response threshold to antigen stimulation.

Binding specificity of multiprotein signaling complexes is determined by both cooperative interactions and affinity preferences.

The generation of multiprotein complexes at receptors and adapter proteins is crucial for the activation of intracellular signaling pathways. In this study, we used multiple biochemical and biophysical methods to examine the binding properties of several SH2 and SH3 domain-containing signaling proteins as they interact with the adapter protein linker for activation of T-cells (LAT) to form multiprotein complexes. We observed that the binding specificity of these proteins for various LAT tyrosines appears to be constrained both by the affinity of binding and by cooperative protein-protein interactions. These studies provide quantitative information on how different binding parameters can determine in vivo binding site specificity observed for multiprotein signaling complexes.